Hospital-acquired bacterial diseases represent a huge block in attempting to care for patients being treated for unrelated medical problems. Of particular concern are multidrug resistant (MDR) pathogens in hospital settings. Acinetobacter baumannii is one such pathogen that has become exceedingly difficult to treat, with the organism having evolved resistance to most commonly used antibiotics during its residence in hospitals. Fluoroquinolones (FQ), which target DNA gyrase and topoisomerase IV, are chief among the antibiotics that have lost usefulness with this organism. The most common resistant A. baumannii strains in hospitals contain three genetic changes, two of which interfere with target binding to FQ, while the third results in overproduction of one of two antibiotic egress pumps. This proposal is directed toward developing new strategies for eliminating fluoroquinolone-resistant (FQR) strains. The central hypothesis of this proposal is that FQR creates points of vulnerability for the organism, and drug targets can be identified that take advantage of this potential Achilles' heel. The proposal seeks to identify drug targets in FQR strains by mapping genetic interactions that protect against stresses caused by the acquired resistance mutations. The proposed studies focus on performing mutant hunts to identify proteins that are required for the survival of FQR bacteria or which, when inactivated, restore the therapeutic usefulness of FQs. The overall strategy involves setting up a bank of parent strains with various combinations of mutations that individually contribute to FQR. Each of these strains is subjected to high-density insertion mutagenesis followed by pooling and analysis by massively parallel sequencing. The fitness of insertions in the FQR backgrounds will be compared to the FQS parent, allowing identification of genes required for viability of each of the strains. Of particular interest in this regard are strains that encode antibiotic egress pumps. The potential for finding a single target that is necessary for survival of strains harboring either of the known pumps that contribute to FQR could allow disruption of a wide spectrum of drug resistant mutants. A similar approach will be taken to identify targets that restore the therapeutic usefulness of FQ. High-density insertion pools in each of the FQR strain backgrounds will be subjected to treatment with the FQ antibiotic ciprofloxacin, and mutants that have low fitness in the presence of the drug relative to the rest of the population will be identified. To demonstrate that the screens identified candidates that are true targets, deletion mutations will be constructed and their ability to reproduce the predicted growth defects will be tested. Long term, the targets identified in this approach will be used in chemical screens to identify novel classes of antimicrobials that target FQR A. baumannii, allowing treatment of drug resistant mutants in hospital settings.